Does propolis have any effect on rheumatoid arthritis? A review study

Abstract Rheumatoid arthritis (RA) is a chronic autoimmune disease in which inflammation and oxidative stress play a key role in its pathophysiology. Complementary therapies along with medications may be effective in the control of RA. Propolis is a natural substance extracted from beehives, which have confirmed anti‐inflammatory and antioxidant effects. The present study aimed to review the possible effects of propolis on inflammation, oxidative stress, and lipid profile in patients with RA. English articles in online databases such as PubMed‑Medline, AMED, Google Scholar, EMBASE, Scopus, and Web of Science databases were searched. Pieces of evidence show that supplementation with propolis may have therapeutic effects on RA patients. Due to increased inflammation and oxidative stress in the affected joints of RA patients, propolis could inhibit the inflammatory cascades by inhibiting the nuclear factor kappa B pathway and reducing reactive oxygen species, malondialdehyde, and interleukin‐17 by increasing some antioxidants. Therefore, inflammation and pain reduce, helping improve and control RA in patients. Further investigations are required with larger sample sizes and different doses of propolis to demonstrate the definite effects of propolis on various aspects of RA.


| INTRODUC TI ON
Rheumatoid arthritis (RA) is a heterogeneous autoimmune and systemic disorder in which cytokines and inflammatory responses play a key role in its pathogenesis (Lubberts & van den Berg, 2013;Nattagh-Eshtivani et al., 2021). Chronic inflammation starts in the synovial membrane and develops into subsequent lesions in the joint cartilage (Lubberts & van den Berg, 2013). The prevalence of RA is estimated at 0.5%-1.0% in the adult population worldwide (Vaghef-Mehrabany et al., 2016). The risk of mortality is higher in patients with RA than in the general population (Helli et al., 2016). Evidence suggests that the higher mortality rate of RA patients is due to the increased cardiovascular risk (Myasoedova & Gabriel, 2010).
Despite the extensive research that has unveiled some of the contributing factors to the initiation and development of RA, the exact etiology of the disease remains unknown (Tobón et al., 2010).
Oxidative stress and inflammation may be significantly involved in the physiopathology of RA, and evidence attests to the increased level of oxidative stress biomarkers and decreased blood antioxidants in patients with RA (Filippin et al., 2008;Kalpakcioglu & Şenel, 2008;Kamanlı et al., 2004;Taysi et al., 2002). Furthermore, it is proposed that reactive oxygen species (ROS) could cause inflammatory responses in RA by activating nuclear factor kappa B (NF-κB) (Filippin et al., 2008). Therefore, using antioxidant supplements may help reduce the symptoms and improve the quality of life in RA patients.
The treatment of RA patients with nonsteroidal anti-inflammatory drugs, glucocorticoids, and disease-modifying antirheumatic drugs could ameliorate the symptoms, although the patients may experience complications such as osteoporosis, diabetes mellitus, and weight gain; these treatments are also rather expensive (Gautam & Jachak, 2009;Mousa et al., 2021;O'Dell, 2004). Therefore, complementary treatments have attracted the attention of researchers to reduce the complications and costs of RA treatment. Studies regarding herbal medicines have confirmed the beneficial effects of medicinal plants on the prevention and management of chronic diseases such as RA (Ernst, 2010;Kaur et al., 2012;Sarker et al., 2020).
Propolis as a complementary medicine has been used in the treatment of various diseases (Farooqui & Farooqui, 2012;Fukuda et al., 2015;Hu et al., 2005;Santos, 2012), and investigations in this regard have confirmed that propolis and its flavones could cause reduction in inflammation (Afsharpour et al., 2017;De Almeida & Menezes, 2002;Jalali et al., 2020). Furthermore, several studies have been conducted on animal models (Table 1). Fang et al. (2013) reported that 160 mg/kg/day of the ethanol extract of propolis (EEP) could significantly decrease IL-6 in mice after 14 weeks of treatment. In another study, Corrêa et al. observed that 100 mg/ kg/day of Brazilian red propolis reduced the IL-6 and TNFα levels in mice after 9 days of administration (Corrêa et al., 2017). The findings of Kismet et al. also demonstrated that the intraperitoneal daily dosage of propolis (200 mg/kg) could significantly decrease TNFα and IL-6 in rats with nonalcoholic fatty liver disease after 2 weeks of treatment (Kismet et al., 2017). In another study, the administration of propolis by gavage (500 mg/kg/day) for 4 days has shown reduction in the intraperitoneal permeability of mice by lowering the effects of inflammatory factors (Lima et al., 2014).
According to the study by Chen et al., propolis gavage (919.5 mg/ kg/day) could decrease serum TNFα, IL-1β, and IL-6, whereas a lower dose (183.9 mg/kg/day) induced moderate responses in terms of TNFα and IL-1β levels . Furthermore, Cheung K.
W. et al. reported that Brazilian propolis and its components (artepillin C) inhibited IL-17 production in human CD4 T cells (Cheung et al., 2011). Therefore, it could be concluded that propolis has antiarthritic effects as T-helper 17 cells, which are involved in the pathogenesis of RA (Iwakura & Ishigame, 2006;Steinman, 2007). Therefore, it is suggested that propolis supplementation in patients with RA could control the disease by decreasing the inflammatory cascade and the secretion of pro-inflammatory indices. Tables 1 and 2 summarize the animal studies and clinical trials regarding the anti-inflammatory effects of propolis, respectively.

| Anti-inflammatory mechanism of propolis
During the inflammation process, macrophages activate and release pro-inflammatory cytokines such as TNFα, IL-1, and IL-6. These F I G U R E 1 Hypothetical mechanism of effects of propolis on reduction of inflammation, oxidative stress, and atherosclerosis activated macrophages induce the translocation of NF-κB. NF-κB activation plays a pivotal role in the production and stimulation of various cytokines and inflammatory mediators (TNFα, IL-1, IL-2, IL-6, and IL-8) while also participating in the regulation of inflammation (Baeuerle, 1991;Surh et al., 2001). Furthermore, NF-κB is critically involved in modulating the survival, differentiation, and activation of immune cells (Liu et al., 2017). The NF-κB signaling pathway also partakes in the production of nitric oxide (NO) by stimulating inducible nitric oxide synthase (iNOS), which is an inflammatory mediator (Pahlavani et al., 2019;Xie et al., 1994).  Abbreviations: ↑, increase; ↓, decrease; ↔, no effect; CRP, C-reactive protein; IL, interleukin; TGFβ, transforming growth factor β; TNFα, tumor necrosis factor-alpha. mediated by NF-κB activation, and the immune response in T cells (Banskota et al., 2001;Paulino et al., 2008). Also as demonstrated in previous research, propolis components could have directly regulated the basic immune cell functions (Wolska et al., 2019). For example, in lipopolysaccharide-stimulated RAW264.7 macrophages, neovestitol, an isoflavonoid derived from propolis, showed an immunological modulatory impact by inhibiting NO production and lowering pro-inflammatory cytokine levels (Bueno- Silva et al., 2017).

TA B L E 2 Summary of clinical trials on anti-inflammatory effects of propolis
Propolis extracts and propolis compounds (caffeic acid, phenethyl ester, quercetin, and hesperidin) could suppress DNA synthesis and the production of inflammatory cytokines (IL-1, IL-12, IL-2, and IL-4) in Th1-and Th2-type T cells while enhancing the production of transforming growth factor-β1 (TGF-β1) (Ansorge et al., 2003).
Furthermore, the suppression of macrophage activation and differentiation has been proposed as one of the possible mechanisms causing propolis' anti-inflammatory and immunological benefits (Araujo et al., 2012).
Evidence suggests that CAPE is a potent modulator of arachidonic acid (AA) that blocks the release of AA from the cell membrane, thereby suppressing the gene expression of lipoxygenase and cyclooxygenase (COX) enzymes (Mirzoeva & Calder, 1996). According to various investigations, CAPE is a dominant and selective inhibitor of NF-κB activation; CAPE has been shown to inhibit NF-κB activation precisely and completely by a wide range of inflammatory stimuli, including TNFα and H 2 O 2 (Ramos & Miranda, 2007).
Propolis also prevents the production of leukotriene and prostaglandin. Propolis flavonoids may be responsible for their effects on the COX enzyme, which has been reported to suppress prostaglandin-endoperoxide synthase (Mirzoeva & Calder, 1996).
In this regard, Woo et al. examined the effects of chrysin on the expression of COX-2, reporting that chrysin could significantly suppress the expression of COX-2 protein and mRNA (Woo et al., 2005). In an in vitro study by Kao et al., the anti-inflammatory effects of artepillin C were investigated on mice, and the obtained results indicated that artepillin C inhibited prostaglandin E2 synthesis and NO production while also reducing NF-κB activity in mice (Kao et al., 2010).
Notably, the anti-inflammatory effects of quercetin have been attributed to the downregulation of the extracellular signalregulated kinase, p38, Akt, Janus kinase-1, tyrosine kinase 2 (TYK2), signal transducer, and NF-κB activator. This compound has also been shown to scavenge free radicals (Kao et al., 2010). with collagen-induced arthritis (Tanaka et al., 2012). Another study found that the anti-inflammatory activity of Brazilian green propolis in stimulated J774A.1 macrophages is mediated through the inhibition of NO and pro-inflammatory cytokines such as TNFα, IL-1, and IL-6 ( Szliszka et al., 2013). As a result, propolis and its ingredients might exert potential natural anti-inflammatory agents that work by modifying immune responses.

| Effects of propolis in relation to oxidative stress in RA
Although the exact etiology of RA remains unknown, several studies have confirmed the role of ROS in the pathophysiology of the disease (Bauerova & Bezek, 2000). ROS are naturally produced during aerobic metabolism, and the cells are protected against ROS by the antioxidant defense system (Roy et al., 2017). When ROS production exceeds the capacity of the antioxidant system, oxidative stress occurs and causes metabolic dysfunction and extensive damage to fats, proteins, and DNA. Ultimately, the free radicals produced from oxygen metabolism destroy the antioxidant system (Tao et al., 2018).
In RA, the activation of neutrophils and macrophages (main cells of inflammatory synovial fluid) increases the production of ROS, which are important mediators of tissue damage in arthritis (Kamanlı et al., 2004;Oztürk et al., 1999). On the contrary, malondialdehyde is altered in the serum of these patients. However, contradictory results have been proposed in this regard Kiziltunc et al., 1998;Sarban et al., 2005).
In addition to acting as a protective mechanism against ROS, antioxidants could suppress the expression of the cytokines and collagenase induced by TNFα, which is also a protective mechanism against arthritis (Halliwell et al., 1988;Sato et al., 1996). It is hypothesized that natural compounds with antioxidant properties may exert protective effects against RA (Bae et al., 2003;Wang et al., 2019).
Propolis is a natural compound that is expected to be effective in reducing oxidative stress levels (Abass et al., 2017;Mujica et al., 2017;Pahlavani et al., 2020). Several studies have demonstrated that propolis could also decrease oxidative stress-related markers (MDA) and increase free radical scavenging enzymes (SOD and GPX) and the total antioxidant capacity (TAC) (Afsharpour et al., 2019;Jasprica et al., 2007). Table 3 presents the summary of the animal studies investigating the effects of propolis on oxidative stress.  According to the literature, the main antioxidant mechanisms of propolis polyphenols may be associated with their scavenging effects on ROS, while nitrogen species and chelating metal ions may also be involved in the production of free radicals, reduction of xanthine oxidase reaction, and synergistic effects with other antioxidants (Kurek-Górecka et al., 2013;Mujica et al., 2017). It is known that phenolic compounds, such as those found in propolis, act as antioxidants by interrupting the chain reaction of lipids (Torel et al., 1986), blocking chemiluminescence processes (Georgetti et al., 2003), and scavenging ROS (Bors et al., 1990).
The antioxidant and reductive capacity of propolis against ROS could be attributed to two main mechanisms, namely the capacity of CAPE in activating NrF2 transcription factor (a regulatory protein associated with antioxidant protection and improvement in antioxidant enzymes) and the phenolic acid and flavonoid contents of propolis (CAPE, quercetin, apigenin, p-coumaric acid, cinnamic acid, and p-vanillin), which neutralize free radicals and oxidant compounds (Ichikawa et al., 2002;Lee et al., 2010). Moreover, propolis has been shown to significantly enhance vitamin C levels in the plasma, kidney, stomach, small intestine, and colon (Seven et al., 2010). Propolis could be absorbed through the bloodstream and act as a hydrophilic antioxidant in the absorption of vitamin C (Seven et al., 2010). Figure 1 represents the hypothetical mechanism of the effects of propolis on the reduction of inflammation and oxidative stress.

| Cardioprotective effects of propolis in RA
Rheumatoid arthritis is an inflammatory disease associated with the increased risk of cardiovascular mortality and morbidity (Aviña-Zubieta et al., 2008;Gonzalez-Gay et al., 2005). However, the exact mechanism of the elevated risk of cardiovascular diseases (CVDs) in RA patients should be further explored. The increased risk of CVD in RA patients may be due to dyslipidemia. Several observational studies have demonstrated that RA is associated with negative effects on lipid profile (Boers et al., 2003;Park et al., 1999Park et al., , 2002. Dyslipidemia causes atherosclerosis and CVD (Nelson, 2013;Tietge, 2014), whereas reduced serum cholesterol leads to a significantly lower risk of CVD (González-Gay & González-Juanatey, 2014; Stamler et al., 2000). Furthermore, inflammation in RA patients plays a pivotal role in disease progression (González-Gay & González-Juanatey, 2014). Scientific evidence suggests that chronic inflammation in patients with RA is associated with a higher risk of CVD (Gonzalez-Gay et al., 2007;Gonzalez-Gay et al., 2005). Inflammation causes oxidative changes, which influence the structure of highdensity lipoprotein (HDL) and decrease apolipoprotein-A1 in RA patients (Charles-Schoeman et al., 2009). In addition, the levels of the antioxidant enzyme associated with HDL (paraoxonase-1) have been reported to be lower in patients with RA compared to healthy controls (Charles-Schoeman et al., 2012). Abbreviations: ↑, increase; ↓, decrease; ↔, no effect; C, control; CAT, catalase; GPX, glutathione peroxidase; GSH, glutathione; GSR, glutathione reductase; GST, glutathione S-transferases; LPO, lipid peroxidation; MDA, malondialdehyde; NO, nitric oxide; NOS, nitric oxide synthases; RNS, reactive nitrogen species; ROS, reactive oxygen species; SOD, superoxide dismutase; T, treatment; TAC, total antioxidant capacity; TBARS, thiobarbituric acid reactive substances.

NATTAGH-ESHTIVANI ET Al.
The cardioprotective effects of propolis have been confirmed in several studies (Ahmed et al., 2017;Alyane et al., 2008;Daleprane & Abdalla, 2013). The in vitro and in vivo studies in this regard have also clarified the molecular mechanisms of these effects, some of which include the improvement in glucose and lipid profiles; reduced activity of scavenger receptors, inflammatory cytokines, and oxidative stress; improvement in endothelial function; and prevention of platelet aggregation (Daleprane & Abdalla, 2013).
Therefore, propolis is considered as an abundant source of polyphenols with a potential role in preventing cardiovascular events.
Propolis has beneficial effects on the regulation of lipid and lipoprotein metabolism. Previous findings have indicated that propolis administration led to reducing liver cholesterol and triglyceride levels and hepatic triglyceride synthesis in rats (Daleprane et al., 2012;Hu et al., 2005). Moreover, treatment with Brazilian propolis in low-density lipoprotein (LDL) receptor knockout mice decreased the levels of triacylglycerol (TAG), total cholesterol (TC), and non-HDL-C (Daleprane et al., 2012). The mice receiving propolis treatment also experienced a significant reduction in TAG and TC, as well as increased HDL-C, compared to the untreated mice. On the same note, Turkish propolis has been reported to prevent alcohol-induced acute liver injury and lipid deposition, exerting positive effects on the lipid profile. Notably, in the mice receiving propolis treatment and alcohol, HDL levels have been reported to be high, and LDL was observed to be lower compared to the mice receiving alcohol only (Kolankaya et al., 2002).
In other studies, propolis has shown favorable effects on the HDL and LDL levels of rats (Hu et al., 2005). For instance, administration of propolis in diabetic rats led to decreased levels of TC, LDL-C, very-low-density lipoprotein (VLDL), and TAG. These findings highlight the role of propolis in the regulation of lipid metabolism, as well as its contribution to the status of lipid abnormalities (Hu et al., 2005). Daleprane  Previous studies have demonstrated the beneficial effects of propolis on the lipid profile (Burdock, 1998;Castaldo & Capasso, 2002;Hu et al., 2005;Munstedt & Zymunt, 2001;Murata et al., 2004;Nader et al., 2010). For instance, Kolankaya et al. conducted an animal study and reported that the EEP at 200 mg/ kg BW/day decreased LDL levels and increased HDL levels in rats (Kolankaya et al., 2002). Consistently, the results of another study indicated that the extracted polyphenols of red propolis significantly lowered TAG and TC and increased HDL-C in the LDL r −/− knockout mice (Daleprane et al., 2012). In a clinical trial conducted by Mujica et al., propolis supplementation for 90 days significantly increased HDL levels and decreased the systolic and diastolic blood pressure, thereby reducing the risk of CVDs (Mujica et al., 2017). The proposed hypocholesterolemic mechanism of propolis is through the protein expression of the ATP-binding cassette (ABC) transporters A1 and G1 (ABCA1 and ABCG1) (Gorinstein et al., 2011).
Various types of propolis could increase ABCA1 gene expression (Ichi et al., 2009;Koya-Miyata et al., 2009), which in turn increases HDL and enhances the cholesterol efflux from the peripheral tissue (Chung et al., 2010;Daleprane et al., 2012;Nader et al., 2010). Therefore, propolis may improve the lipid profile by upregulating ABCA1 gene expression. In addition, the ethanol extract of Brazilian red propolis has been reported to increase the ABCA1 promoter activity in THP-1 macrophages (Iio et al., 2012). Given that patients with RA have impaired ATP-binding cassette G1-mediated CEC due to the disease activity and its complications (Ronda et al., 2014), propolis supplementation may effectively decrease the disease symptoms, thereby decreasing the risk of CVDs. Simultaneously with the increase in the ABCA1 cassette, Brazilian red propolis could upregulate ApoA-1, which is involved in the cholesterol efflux by macrophages. The effects of propolis on ABCA1 could be attributed to the activation of PPARγ and LXRα (Iio et al., 2012). Table 4  Nitric oxide is an endothelium-derived relaxing factor with vasodilatory and antiaggregative properties, which protects the blood vessels at low concentrations. However, the excessive NO produced by inflammatory cells may react with other nitrogen and oxygen species and stimulate oxidative stress (Ali et al., 2014). Several studies have reported increased NO levels in the serum of patients with RA (Ali et al., 2014;Ersoy et al., 2002;Mahmoud & Ismail, 2011).
According to an animal study, propolis intake in diabetic mice resulted in the reduction of NO and NOS levels (Hu et al., 2005). Propolis reduces NO levels by decreasing NOS activity, thereby protecting the endothelial cells of the blood vessels. Furthermore, the EEP could prevent NO production by reducing iNOS expression in Raw 264.7 macrophages and inhibiting the catalytic activity of iNOS. On the contrary, excessive NO production is involved in the cardiovascular inflammatory process, and propolis may affect the regulation of NO levels through its anti-inflammatory activities.

| Strengths and limitations
This review study aimed to assess the effects of propolis on inflammation, oxidative stress, and cardiometabolic indices in RA patients.
The main limitation of our study was the heterogeneity of the reported data in the reviewed studies, and quality assessment of the studies might have led to more accurate results for the generalization of the data. To the best of our knowledge, this is the first review study that has gathered an in-depth scientific demonstration of the possible effects of propolis on patients with RA.

| CON CLUS ION
The present study suggested that propolis may have beneficial effects on oxidative stress biomarkers and inflammation process in RA patients due to its potent antioxidant and polyphenolic properties. Further studies particularly clinical trials must be conducted to TA B L E 4 Summary of animal studies on effects of propolis on lipid profile Abbreviations: ↑, increase; ↓, decrease; ↔, no effect; C, control; HDL, high-density lipoprotein; LDL, low-density lipoprotein; T, treatment; TC, total cholesterol; TG, triglyceride.
demonstrate the definitive effects of propolis on multiple aspects of RA disease.

ACK N OWLED G M ENTS
The authors thank Dr. NaeimRoshan for editing the manuscript.

CO N FLI C T S O F I NTE R E S T
The authors also declare that they have no conflict of interest.

E TH I C A L A PPROVA L
No ethical approval was required, as this is a review article with no original research data.

I N FO R M E D CO N S E NT
There were no study participants in this review article, and informed consent was not required.

DATA AVA I L A B I L I T Y S TAT E M E N T
All the data used in this study can be made available on reasonable request.